The present invention relates to the novel proteins termed AURORA ONE and AURORA TWO (xe2x80x9cAUR1 and AUR2xe2x80x9d), nucleotide sequences encoding AUR1 and/or AUR2, as well as various products and methods useful for the diagnosis and treatment of various AUR1 and/or AUR2 related diseases and conditions.
The following description of the background of the invention is provided to aid in understanding the invention but is not admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
The best characterized protein kinases in eukaryotes phosphorylate proteins on the alcohol moiety of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as xe2x80x9cdual specificityxe2x80x9d kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues.
Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
The serine/threonine kinase family includes members found at all steps of various signaling cascades, including those involved in controlling cell growth, migration, differentiation and secretion of hormones, phosphorylation of transcription factors resulting in altered gene expression, muscle contraction, glucose metabolism, control of cellular protein synthesis, and regulation of the cell cycle.
Chromosomal abnormalities are a hallmark of human cancer, reflecting the deleterious consequences of the gain or loss of genetic information (Mitelman et al., Nature Genet. 15:417-474, 1997; Hartwell et al., Science 266:1821-1828, 1994). Some of these defects may have a causal role in cellular transformation due to loss of a negative growth regulator, loss of a gene responsible for maintenance of genome integrity, or through the amplification or activation of an oncogene (Kinzler et al., Nature 386:761-763, 1997; Hunter Cell 88:333-346, 1997). Alternatively, these abnormalities may be a consequence of tumor progression where mitotic checkpoints have been disrupted, resulting in abnormal nuclei, miss-segregated chromosomes, and aneuploidy (Elledge Science 274:1664-1672, 1996; Sherr Science 274:1672-1677, 1996).
The present invention relates in part to AUR1 and/or AUR2 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. The utility of the present invention includes the ability to screen for inhibitors of cell growth and to develop small molecule therapeutics for treating cancers.
Thus, in a first aspect, the invention features an isolated, enriched, or purified nucleic acid encoding an AUR1 and/or AUR2 polypeptide.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant a polymer of 6 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA and RNA that is isolated from a natural source or that is synthesized. In certain embodiments of the invention, longer nucleic acids are preferred, for example those of 300, 600, 900 or more nucleotides and/or those having at least 50%, 60%, 75%, 90%, 95% or 99% identity to the full length sequence shown in SEQ ID NO:1 or SEQ ID NO:2. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
By xe2x80x9can AUR1 and/or AUR2 polypeptidexe2x80x9d is meant 25 (preferably 30, more preferably 35, most preferably 40) or more contiguous amino acids set forth in the full length amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, or a functional derivative thereof as described herein. In certain aspects, polypeptides of 100, 200, 300 or more amino acids are preferred. The AUR1 and/or AUR2 polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained.
Also included are inactive and activated mutants of AUR1 and/or AUR2, including, but not limited to those defined in Example 11 herein. By xe2x80x9cinactivexe2x80x9d is meant an AUR1 and/or AUR2 polypeptide which lacks kinase activity. In some embodiments, the essential lysine (residue 162) is mutated. Preferably the polypeptide is otherwise unchanged. By xe2x80x9cactivatedxe2x80x9d is meant an AUR1 and/or AUR2 polypeptide which has kinase activity in vitro, preferably in situations where the unmutated polypeptide does not. Preferably, the AUR1 and/or AUR2 polypeptide is mutated to mimic constitutive phosphorylation. In some embodiments, the threonine at residue 288 in the activation loop is modified to an aspartic acid.
The amino acid sequence will be substantially similar to the sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or fragments thereof. A sequence that is substantially similar will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:3 or SEQ ID NO:4.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
In a preferred embodiment, the invention features a nucleic acid molecule comprising a nucleotide sequence that: (a) encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleic acid molecule of (a) and encodes a naturally occurring AUR1 and/or AUR2 polypeptide; (d) encodes AUR1 and/or AUR2 polypeptide having the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of the following segments of amino acid residues: 1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or 275-403 of SEQ ID NO:4; (e) is the complement of the nucleotide sequence of (d); (f) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid residues 1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, 275-403 of SEQ ID NO:4; (g) is the complement of the nucleotide sequence of (f); (h) encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of the domains selected from the group consisting of a C-terminal domain, a catalytic domain, and an N-terminal domain; or (i) is the complement of the nucleotide sequence of (h).
The term xe2x80x9ccomplementxe2x80x9d refers to two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. A nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
The term xe2x80x9cdomainxe2x80x9d refers to a region of a polypeptide which contains a particular function. For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
The term xe2x80x9cN-terminal domainxe2x80x9d refers to a portion of the full length amino acid sequence spanning from the amino terminus to the start of the catalytic domain. The N-terminal domain spans amino acid residues 1-73 of the sequence set forth in SEQ ID NO:3 or amino acids 1-130 of the sequence set forth in SEQ ID NO:4.
The term xe2x80x9ccatalytic domainxe2x80x9d refers to a portion of the full length amino acid sequence that does not contain the N-terminal domain or the C-terminal domain and has catalytic activity. The catalytic domain spans amino acid residues 73-271 of the sequence set forth in SEQ ID NO:3 or residues 130-274 of the sequence set forth in SEQ ID NO:4.
The term xe2x80x9cC-terminal domainxe2x80x9d refers to a portion of the full length amino acid sequence that begins at the end of the catalytic domain and ends at the carboxyl terminal amino acid, which is the last amino acid encoded before the stop codon in the nucleic acid sequence. The C-terminal domain spans amino acid residues 272-344 of the sequence set forth in SEQ ID NO:3 or amino acids 275-403 of the sequence set forth in SEQ ID NO:4.
In preferred embodiments, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, encodes the full length amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, a functional derivative thereof, or at least 25, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids thereof. The AUR1 and/or AUR2 polypeptide comprises, consists essentially of, or consists of at least 25, 30, 35, or 40 contiguous amino acids of an AUR1 and/or AUR2 polypeptide. The nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization. The natural source may be mammalian, preferably human, blood, semen, or tissue and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
In yet other preferred embodiments, the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides. Examples of amino acid sequences of the present invention include the following amino acid sequences (the isolated, purified or enriched nucleic acids encoding them are also within the scope of the present invention): ENSYPWPYGRQ (SEQ ID NO:5), CISGP (SEQ ID NO:6), QFPQ (SEQ ID NO:7), VNSGQ (SEQ ID NO:8), RKEPVTPSA-LV (SEQ ID NO:9), LMSRSNVQPTAAP (SEQ ID NO:10), VQNQKQKQLQATSVPH (SEQ ID NO:11), PVSRPLNNTQK (SEQ ID NO:12), VMENSSGTPD (SEQ ID NO:13), ILTRHFTID (SEQ ID NO:14),. and SKQPLPSAPENNPEEQLASKQK (SEQ ID NO:15).
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, are meant regions present on two or more nucleic acids encoding an AUR1 and/or AUR2 polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding AUR1 and/or AUR2 polypeptides are provided in Abe et al. J. Biol. Chem. 19:13361-13368, 1992 (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d is meant a sequence present in a full length nucleic acid coding for an AUR1 and/or AUR2 polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 30 to 45 contiguous nucleotides present in the full length nucleic acid encoding an AUR1 and/or AUR2 polypeptide. In particular, a unique nucleic acid region is preferably of mammalian origin.
In a preferred embodiment, the isolated, enriched or purified nucleic acid molecule encoding AUR1 and/or AUR2 polypeptide, comprises a vector or promoter effective to initiate transcription in a host cell.
The invention also features a nucleic acid probe for the detection of nucleic acid encoding an AUR1 and/or AUR2 polypeptide in a sample. The nucleic acid probe contains a nucleotide base sequence that will hybridize to a sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or a functional derivative thereof.
In preferred embodiments the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 or a functional derivative thereof. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
By stringent hybridization assay conditions is meant hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5xc3x97SSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5xc3x97Denhart solution at 42xc2x0 C. overnight; washing with 2xc3x97SSC, 0.1% SDS at 45xc2x0 C.; and washing with 0.2xc3x97SSC, 0.1% SDS at 45xc2x0 C.
Methods for using the probes include detecting the presence or amount of AUR1 and/or AUR2 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to AUR1 and/or AUR2 RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for an AUR1 and/or AUR2 polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding an AUR1 and/or AUR2 polypeptide and a transcriptional termination region functional in a cell.
In another aspect, the invention describes a recombinant cell or tissue containing nucleic acid coding for an AUR1 and/or AUR2 polypeptide. In such cells, the nucleic acid may be under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the AUR1 and/or AUR2 polypeptide.
The polypeptide is preferably a fragment of the protein encoded by the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. By xe2x80x9cfragment,xe2x80x9d is meant an amino acid sequence present in a full-length AUR1 and/or AUR2 polypeptide that is not present in any other naturally occurring polypeptide. Preferably, such a sequence comprises 6 contiguous amino acids present in the full sequence. More preferably, such a sequence comprises 12 contiguous amino acids present in the full sequence. Even more preferably, such a sequence comprises 18 contiguous amino acids present in the full sequence.
In another aspect the invention features an isolated, enriched, or purified AUR1 and/or AUR2 polypeptide.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 2 (preferably 7, more preferably 13, most preferably 25) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. In certain aspects longer polypeptides are preferred, such as those with 402, 407, 413, or 425 contiguous amino acids set forth in SEQ ID NO:3 or SEQ ID NO:4. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-5 fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In preferred embodiments, the AUR1 and/or AUR2 polypeptide contains at least 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, or a functional derivative thereof.
Also included are inactive and activated mutants of AUR1 and/or AUR2, including, but not limited to those defined in Example 11 herein. By xe2x80x9cinactivexe2x80x9d is meant an AUR1 and/or AUR2 polypeptide which lacks kinase activity. In some embodiments, the essential lysine (residue 162) is mutated. Preferably the polypeptide is otherwise unchanged. By xe2x80x9cactivatedxe2x80x9d is meant an AUR1 and/or AUR2 polypeptide which has kinase activity in vitro, preferably in situations where the unmutated polypeptide does not. Preferably, the AUR1 and/or AUR2 polypeptide is mutated to mimic constitutive phosphorylation. In some embodiments, the threonine at residue 288 in the activation loop is modified to an aspartic acid.
The polypeptide may be isolated from a natural source by methods well-known in the art. The natural source may be mammalian, preferably human, blood, semen,. or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer.
In a preferred embodiment, the invention features a polypeptide comprising an amino acid sequence having (a) the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b) the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of the following segments of amino acid residues: 1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or 275-403 of SEQ ID NO:4; (c) the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid residues 1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or 275-403 of SEQ ID NO:4; or (d) the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of the domains selected from the group consisting of a C-terminal domain, a catalytic domain, and an N-terminal domain.
In some embodiments the invention includes a recombinant AUR1 and/or AUR2 polypeptide. By xe2x80x9crecombinant AUR1 and/or AUR2 polypeptidexe2x80x9d is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
In yet another aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to an AUR1 and/or AUR2 polypeptide or an AUR1 and/or AUR2 polypeptide domain or fragment. By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to the target (AUR1 and/or AUR2) polypeptide with greater affinity than it binds to other polypeptides under specified conditions. Antibodies or antibody fragments are polypeptides which contain regions that can bind other polypeptides. The term xe2x80x9cspecific binding affinityxe2x80x9d describes an antibody that binds to an AUR1 and/or AUR2 polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110).
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a. portion of an antibody, often the hyper variable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hyper variable region is a portion of an antibody that physically binds to the polypeptide target.
Antibodies or antibody fragments having specific binding affinity to an AUR1 and/or AUR2 polypeptide may be used in methods for detecting the presence and/or amount of AUR1 and/or AUR2 polypeptide in a sample by probing the sample with the antibody under conditions suitable for AUR1 and/or AUR2-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the AUR1 and/or AUR2 polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for AUR1 and/or AUR2 as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
An antibody or antibody fragment with specific binding affinity to an AUR1 and/or AUR2 polypeptide can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
Antibodies having specific binding affinity to an AUR1 and/or AUR2 polypeptide may be used in methods for detecting the presence and/or amount of AUR1 and/or AUR2 polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the AUR1 and/or AUR2 polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
In another aspect, the invention features a hybridoma which produces an antibody having specific binding affinity to an AUR1 and/or AUR2 polypeptide or an AUR1 and/or AUR2 polypeptide domain. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody, for example an antibody to AUR1 and/or AUR2. In preferred embodiments the antibody to AUR1 and/or AUR2 comprises a sequence of amino acids that is able to specifically bind an AUR1 and/or AUR2 polypeptide.
In another aspect, the invention features an AUR1 and/or AUR2 polypeptide binding agent able to bind to an AUR1 and/or AUR2 polypeptide. The binding agent is preferably a purified antibody which recognizes an epitope present on an AUR1 and/or AUR2 polypeptide. Other binding agents include molecules which bind to the AUR1 and/or AUR2 polypeptide and analogous molecules which bind to an AUR1 and/or AUR2 polypeptide. Such binding agents may be identified by using assays that measure AUR1 and/or AUR2 binding partner activity, such as those that measure PDGFR activity.
The invention features a method for screening for human cells containing an AUR1 and/or AUR2 polypeptide or an equivalent sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying AUR1 and/or AUR2 (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
In another aspect, the invention provides a method for identifying a substance capable of modulating AUR1 and/or AUR2 activity comprising the steps of (a) contacting AUR1 and/or AUR2 polypeptide with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.
The term xe2x80x9cmodulatesxe2x80x9d refers to the ability of a compound to alter the function of AUR1 and/or AUR2. A modulator preferably activates or inhibits the activity of AUR1 and/or AUR2 depending on the concentration of the compound exposed to AUR1 and/or AUR2.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the cellular activity of AUR1 and/or AUR2. The term xe2x80x9cinhibitxe2x80x9d refers to decreasing the cellular activity of AUR1 and/or AUR2. AUR1 and/or AUR2 activity is preferably the interaction with a natural binding partner.
The term xe2x80x9cmodulatesxe2x80x9d also refers to altering the function of AUR1 and/or AUR2 by increasing or decreasing the probability that a complex forms between AUR1 and/or AUR2 and a natural binding partner. A modulator preferably increases the probability that such a complex forms between AUR1 and/or AUR2 and the natural binding partner, more preferably increases or decreases the probability that a complex forms between AUR1 and/or AUR2 and the natural binding partner depending on the concentration of the compound exposed to AUR1 and/or AUR2, and most preferably decreases the probability that a complex forms between AUR1 and/or AUR2 and the natural binding partner.
The term xe2x80x9ccomplexxe2x80x9d refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
The term xe2x80x9cnatural binding partnerxe2x80x9d refers to polypeptides or nucleic acids that bind to AUR1 and/or AUR2 in cells. A change in the interaction between AUR1 and/or AUR2 and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of AUR1 and/or AUR2/natural binding partner complex.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods. The solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
In another aspect, the invention provides for the treatment of diseases by administering to a patient in need of such treatment a substance that modulates the activity of AUR1 and/or AUR2. Such substances preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in example 13 below). Examples of substances that can be screened for favorable activity are provided in section XI below. The diseases that could be treated by a modulator of AUR1 and/or AUR2 activity preferably include colon, breast, renal, ovarian, bladder, head and neck cancers, and gliomas, medulloblastomas, chondrosarcomas, and pancreatic tumors, and preferably include breast, colon, and renal cancers, and more preferably, colon cancer. The substances that modulate the activity of AUR1 and/or AUR2 preferably include, but are not limited to, antisense oligonucleotides, as described herein, and inhibitors of protein kinases, as determined by methods and screens described herein in the Examples.
Another aspect of the invention features a method for detection of aur1 and/or aur2 in a sample as a diagnostic tool for diseases comprising the steps of (a) contacting said sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of aur1 and/or aur2, said probe comprising the nucleic acid sequence encoding an AUR1 and/or AUR2 polypeptide, a fragment thereof, or the complement of said sequence or fragment; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of said disease.
The aur1 and/or aur2 xe2x80x9ctarget regionxe2x80x9d is the nucleotide base sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, a functional derivative thereof, or a fragment thereof to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the aur1 and/or aur2 target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
In preferred embodiments the nucleic acid probe hybridizes to an aur1 and/or aur2 target region encoding at least 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 or a functional derivative thereof. Hybridization conditions should be such that hybridization occurs only with aur1 and/or aur2 in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
The diseases for which detection of aur1 and/or aur2 in a sample could be diagnostic include diseases in which aur1 and/or aur2 nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells. By xe2x80x9camplificationxe2x80x9d is meant increased numbers of aur1 and/or aur2 DNA or RNA in a cell compared with normal cells. In normal cells, aur1 and aur2 are found as single copy genes. In selected diseases, the chromosomal location of aur1 and/or aur2 is amplified, resulting in multiple copies of the gene, or amplification. Gene amplification can lead to amplification of aur1 and/or aur2 RNA, or aur1 and/or aur2 RNA can be amplified in the absence of aur1 and/or aur2 DNA amplification.
xe2x80x9cAmplificationxe2x80x9d as it refers to RNA can be the detectable presence of aur1 and/or aur2 RNA in cells, since in some normal cells there is no basal expression of aur1 and/or aur2 RNA. In other normal cells, a basal level of expression of aur1 and/or aur2 exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, aur1 and/or aur2 RNA, compared to the basal level.
The diseases that could be diagnosed by detection of aur1 and/or aur2 in a sample preferably include colon, breast, renal, ovarian, bladder, head and neck cancers, and gliomas, medulloblastomas, chondrosarcomas, and pancreatic tumors, and preferably include breast, colon, and renal cancers, and more preferably, colon cancer.
The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
Another aspect of the invention features antisense oligonucleotides to the nucleic acid sequences encoding AUR1 and/or AUR2 polypeptides contained in SEQ ID NO:1 and/or SEQ ID NO:2, and fragments thereof. In a preferred invention the antisense oligonucleotides are synthesized as phosphorothionates. In a preferred embodiment the antisense oligonucleotides comprise the following sequences 5xe2x80x2-3xe2x80x2: nucleotides 1743-1763 of aur2: CAGGGCAGAGTGGTCACTTTC (SEQ ID NO:30), nucleotides 42-62 of aur2: CGTCCGCCACTCCGACCAGCC (SEQ ID NO:31), nucleotides 1654-1674 of aur2: TGCAGTCGAACCTTGCCTCCA (SEQ ID NO:32).
The antisense oligonucleotides of the invention are preferably used to inhibit AUR1 and/or AUR2 protein expression in vivo in normal and tumor cells. Antisense oligonucleotides can be used either singly or in combination. In a preferred embodiment, either SEQ ID NO:30 and SEQ ID NO:32 or SEQ ID NO:31 and SEQ ID NO:32 are used jointly. In a preferred embodiment, expression of AUR2 is significantly reduced, and more preferably reduced to below the limit of detection. In other preferred embodiments, treatment with SEQ ID NO:31 and SEQ ID NO:32 inhibits growth and/or induces apoptosis in cells. Antisense oligonucleotides can also be used to inhibit AUR1 and/or AUR2 protein expression in human tumor cell xenografts in nude mice. Antisense oligonucleotides may preferentially be used as a treatment in various human tumors over expressing AUR2.
Additional antisense oligonucleotides and effective combinations can be identified by methods well known in the art. Briefly, cells or tissues over expressing aur1 and/or aur2 can be contacted with antisense oligonucleotides, either singly or in combination, and the expression of aur1 and/or aur2 RNA, and/or AUR1 and/or AUR2 polypeptide can be determined by methods described herein. Preferably, treatment with aur1 and/or aur2 causes a decrease in the expression of aur1 and/or aur2 RNA and/or AUR1 and/or AUR2 polypeptide, more preferably expression is decreased significantly (1 to 2-fold), most preferably expression is decreased to an undetectable level.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.